Ubiquitination plays a central role in regulating protein turnover. Ubiquitin is a highly conserved 76-amino acid protein and one of the most abundant proteins in cells. Ubiquitin proteins are found linked to other proteins as post-translational modification. Polyubiquitination, attachment of more than 4 ubiquitins has been shown to direct proteins for degradation. Ubiquitin conjugation requires sequential action of three enzymes or protein complexes, namely ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). Ubiquitin is initially attached to E1 enzyme in an ATP dependent reaction. After ubiquitin activation, it is transferred to the E2 enzyme via thiolester linkage. Finally, the E3 ligase enzyme brings together the substrate and the E2 with attached ubiquitin. Once a polyubiquitin chain is assembled on a substrate, the substrate is captured and degraded by 26S proteasomes. The step of substrate recognition is mediated by E3 ligases, which typically contain a RING domain as a ‘docking site’ for E2s. The E3 ligase proteins appear in two forms: as single chain E3s that bind directly E2 and their substrate, and as multisubunit complexes in which a RING finger protein is an essential component. The classification of E3 ligases is based on the presence of domains like E6-AP C-terminus, U-box or the Really Interesting Gene (RING) domain. The RING domain is a particular type of Zinc-finger domain that binds two zinc atoms and that is involved in protein-protein interactions. Ubiquitin dependent protein degradation has been acknowledged as an important regulatory means of cell cycle, DNA repair, transcription, protein quality control and immune response.
The hub1 (histone monoubiquitination 1) mutant, also known as hub1-1, ang4-1 or rdo4, belongs to the angusta class of recessive mutants characterised by a reduced leaf size and narrow lamina (Berná et al., Genetics 152, 729-742, 1999), and reduced seed dormancy (Peeters et al., Physiol. Plant. 115, 604-612, 2002; Liu et al., Plant Cell 19, 433-444, 2007). Germination tests (wherein the percentage of germinated seeds was scored after 7 days) showed that this percentage was higher compared to the wild type seeds. However, seedling growth was dependent on the presence of sucrose in the medium (Liu et al., 2007). The mutant plants are small compared to wild type and have narrow leaves, and also root growth is negatively affected (Fleury et al., Plant Cell 19, 417-432, 2007). Overexpression of the HUB1 gene under control of the strong CaMV35S promoter in Arabidopsis resulted in plants having increased leaf size (Cnops et al., WO 2006/027310). It is postulated (Liu et al., 2007 and Fleury et al., 2007) that HUB1 is involved chromatin remodelling, but HUB1 may also be involved in protein degradation: BRE1, the yeast homologue of HUB1, is shown to interact with RAD6 and the proteasome (Xiao et al., Mol. Cell. Biol. 25, 637-651, 2005) and HUB1 or HUB2 furthermore aligns with Staring, a protein which is also involved in protein degradation (Chin et al., J. Biol. Chem. 277, 35071-35079, 2002).